*4.1.1 Attachment and entry of the virus into the host cell*

The first phase of the virus replication cycle is the attachment and entry of the virus into the host cell. There are two ways through which SARS-CoV-2 can gain entry into host cells. The first is through the binding interaction of SARS-COV-2 spike protein (S) with the ACE2 receptor and transmembrane protease serine 2 of the target cell (TMPRSS2), while the second is through endocytosis [28]. ACE2 is the coupling site for SARS-CoV-2 spike protein, while TMPRSS2 facilitates host cell entrance; therefore, some drugs that interfere with ACE2 or TMPRSS2 have the potential for being repurposed in the management of COVID-19 because they will prevent the entry of SARS-CoV-2 into host cells. In theory, drugs such as dexamethasone, estradiol, isotretinoin, retinoic acid, and spironolactone can influence the expression of ACE2, while drugs such as bicalutamide, bromhexine, camostat mesylate, and nafamostat act similarly with TMPRSS2 [28]. In an experiment involving SARS-CoV-2 spike pseudotyped virus, dexamethasone was found to bind to ACE2, preventing binding of the spike protein of the virus to ACE2 and preventing entry of the

virus into the target cell [29]. This is the likely mechanism by which dexamethasone would find activity against SARS-CoV-2 as a repurposed drug. Similarly, bromhexine is a TMPRSS2 protease blocker that prevents viropexis of SARS-CoV-2 into target cells through its blocking effect on TMPRSS2 [28].

Viral attachment and entry into the host cell involve the attachment of spike protein of the SARS-CoV-2 to ACE2 and further involvement of TMPRSS2. If potential drugs for repurposing in COVID-19 treatment leverage their possible effects on ACE2 and TMPRSS2, then drugs that could influence the viral strike protein are also theoretically useful enough to be repurposed in COVID-19 treatment. One such drug is bamlanivimab.

Bamlanivimab is a recombinant human IgG1κ monoclonal antibody with activity against the spike (S) surface protein of SARS-CoV-2 itself, not the ACE2 or TMPRSS2 of the host cell [28]. Umifenovir and nelfinavir are antiviral medications. Umifenovir was previously used in the prophylaxis and management of influenza, while nelfinavir is an antiretroviral medication used in HIV. Umifenovir inhibits spike protein trimerization, while nelfinavir inhibits membrane fusion. This implies that both drugs have the potential for repurposing in COVID-19 management [28].

Endocytosis is a process through which cells take in foreign material by enveloping it with their membrane [29]. Research has shown that SARS-CoV-2 is capable of entering host cells by means of endocytosis. Drugs that prevent the entry of SARS-CoV-2 by endocytosis into the host cell can be repurposed in COVID-19. Such drugs include chloroquine, hydroxychloroquine, artemisinin, amodiaquine, chlorpromazine, niclosamide, imatinib, artesunate, baricitinib, verapamil, and amiodarone [28]. Many of these drugs work by inhibiting membrane fusion between SARS-CoV-2 spike protein and the host cell membrane.

#### *4.1.2 Chloroquine and hydroxychloroquine*

Chloroquine belongs to the chemical class of antimalarial medications called 4-aminoquinolines [30, 31]. Hydroxychloroquine (HCQ ) is a derivative of chloroquine obtained by β-hydroxylation of the N-ethyl substituent of chloroquine to give hydroxychloroquine, which has a hydroxyl group at the end of the side chain. Chloroquine was known to be an effective drug in the treatment of malaria due to its high activity against the asexual erythrocytic forms of the plasmodium. This high profile of effectiveness was however affected negatively due to the growing cases of plasmodial resistance to available antimalarial agents. Although chloroquine was initially developed to treat malaria, the focus has largely shifted to its antirheumatic and antiviral activity. Over the past 20 years, a great deal of research has gone into the investigation of the antiviral effects of chloroquine [32].

As an antimalarial agent, chloroquine/hydroxychloroquine enters the feeding vacuoles of the malaria parasite where it prevents the conversion of heme (a toxic product of the breakdown of hemoglobin) to hemozoin (which is not harmful to the parasite). Accumulation of heme leads to the death of the malaria parasite.

As a repurposed drug for COVID-19, in order to prevent the fusion of SARS-CoV-2 with the host cell membranes, chloroquine/hydroxychloroquine is thought to work by blocking endocytic proteins and elevating the pH of the endosomes [33]. The endocytic pathway interference, sialic acid receptor blockage, limitation of pH-mediated spike (S) protein cleavage at the angiotensin-converting enzyme 2 (ACE2) binding site, and cytokine storm prevention are all part of the mechanism

of action of chloroquine/hydroxychloroquine [34]. The major drawbacks of the use of chloroquine or hydroxychloroquine in COVID-19 treatment are adverse effects such as retinopathy, prolonged QT interval on the ECG, and cardiotoxicity [35]. Hydroxychloroquine is preferred to chloroquine due to its increased hydrophilic nature and decreased toxicity. Hydroxychloroquine is also better tolerated than chloroquine [36].

In recent years, rheumatoid arthritis, lupus erythematosus, and amoebic hepatitis have all been managed with chloroquine and its hydroxyl derivative, hydroxychloroquine as anti-inflammatory agents. Chloroquine exhibits potent antiviral action against a variety of DNA and RNA viruses, including HIV-1, Influenza A, Influenza B, Coronavirus (SARS-CoV2), and many more. Recent reports and published research revealed that chloroquine and hydroxychloroquine were linked to slowed COVID-19 progression and shorter symptom duration. In June 2020, however, FDA revokes the emergency use authorization of the use of both chloroquine and hydroxychloroquine in the management of COVID-19, thus discouraging its use [32, 33].

### *4.1.3 Viral replication*

After attachment and entry of the virus into the host cell, the SARS-CoV-2 life cycle then proceeds to the release of the viral RNA genome into the cytoplasm and translation of the replicase genes, which develop the replicase transcriptase complex (RTC) [28]. RNA replication is carried out by RNA-dependent RNA polymerase RdRp, which is incorporated within the RTC [28, 37]. Favipiravir, tenofovir, sofosbuvir, clevudine, and a number of other drugs have been suggested for COVID-19 repurposing due to their inhibitory effect on RdRp [28, 38]. Remdesivir (in its active form) is a nucleoside analog that inhibits the SARS-CoV-2 RdRp thereby preventing further replication of SARS-CoV-2 [39]. Other RNA replication inhibitors include ivermectin, mefloquine, doxycycline, emtricitabine, and tacrolimus.

After viral RNA replication, comes the translation of viral structural proteins. Atazanavir, saquinavir, lopinavir, and ritonavir were considered repurposing candidates for COVID-19 based on their activity as protease inhibitors (just as they are HIV protease inhibitors). This phase involves proteolytic processing of viral proteins, thus the use of protease inhibitors to interfere with and prevent the translation of proteins [28].

### *4.1.4 Viral assembly and release*

This phase comprises of formation of mature virions and exocytosis. Progeny viruses are assembled in the endoplasmic reticulum-Golgi intermediate complex following the synthesis and processing of the viral structural proteins and are then transported in vesicles to be released by exocytosis. Candidates for repurposing include antiviral medications that target this phase of SARS-CoV-2 replication. Oseltamivir and daclatasvir are such medications [28]. Daclatasvir inhibits viral assembly while oseltamivir inhibits virus release. Oseltamivir interacts with exocytosis-related elements, preventing the viral escape from the cell [40]. Oseltamivir is effective for a number of avian influenza virus strains and functions as a neuraminidase inhibitor against the influenza virus [41].
